Key Points• JAK3-mediated phosphorylation of EZH2 resulted in EZH2 oncogenic function independent of its enzymatic activity.• Targeted inhibition of JAK3 may be a promising treatment in NK/TL through suppressing noncanonical EZH2 activity.The best-understood mechanism by which EZH2 exerts its oncogenic function is through polycomb repressive complex 2 (PRC2)-mediated gene repression, which requires its histone methyltransferase activity. However, small-molecule inhibitors of EZH2 that selectively target its enzymatic activity turn out to be potent only for lymphoma cells with EZH2-activating mutation. Intriguingly, recent discoveries, including ours, have placed EZH2 into the category of transcriptional coactivators and thus raised the possibility of noncanonical signaling pathways. However, it remains unclear how EZH2 switches to this catalytic independent function. In the current study, using natural killer/T-cell lymphoma (NKTL) as a disease model, we found that phosphorylation of EZH2 by JAK3 promotes the dissociation of the PRC2 complex leading to decreased global H3K27me3 levels, while it switches EZH2 to a transcriptional activator, conferring higher proliferative capacity of the affected cells. Gene expression data analysis also suggests that the noncanonical function of EZH2 as a transcriptional activator upregulates a set of genes involved in DNA replication, cell cycle, biosynthesis, stemness, and invasiveness. Consistently, JAK3 inhibitor was able to significantly reduce the growth of NKTL cells, in an EZH2 phosphorylation-dependent manner, whereas various compounds recently developed to inhibit EZH2 methyltransferase activity have no such effect. Thus, pharmacological inhibition of JAK3 activity may provide a promising treatment option for NKTL through the novel mechanism of suppressing noncanonical EZH2 activity. (Blood. 2016;128(7):948-958)
Stat3 is a member of the signal transducer and activator of transcription family, which is important for cytokine signaling as well as for a number of cellular processes including cell proliferation, anti-apoptosis and immune responses. In recent years, evidence has emerged suggesting that Stat3 also participates in cell invasion and motility. However, how Stat3 regulates these processes remains poorly understood. Here, we find that loss of Stat3 expression in mouse embryonic fibroblasts leads to an elevation of Rac1 activity, which promotes a random mode of migration by reducing directional persistence and formation of actin stress fibers. Through rescue experiments, we demonstrate that Stat3 can regulate the activation of Rac1 to mediate persistent directional migration and that this function is not dependent on Stat3 transcriptional activity. We find that Stat3 binds to βPIX, a Rac1 activator, and that this interaction could represent a mechanism by which cytoplasmic Stat3 regulates Rac1 activity to modulate the organization of actin cytoskeleton and directional migration.
The interleukin (IL)-6-type cytokines play major roles in a variety of biological processes by signaling through a common receptor subunit, glycoprotein (gp) 130. We performed yeast two-hybrid screening to identify new binding partners of the activated gp130 and the associated Janus kinases. LMO4, a LIM domain-containing protein that belongs to a family of oncogenes, was identified in this assay. Further studies show that LMO4 associates with gp130 and Janus kinase1 in several mammalian cell types. It also interacts with proteintyrosine phosphatase 2 (SHP2) and suppressor of cytokine signaling 3 (SOCS3). The binding domains involved in these interactions were mapped, and the interactions were shown to be in a direct manner by in vitro binding assays. It is likely that LMO4 exists in the gp130 complex. The cellular localization of LMO4 was detected primarily in the nucleus with a substantial amount also detected in the cytoplasm in several cell types. The interleukin (IL 1 )-6-type cytokines belong to a cytokine family that plays major roles in hematopoiesis, immune responses, inflammation, and neural development by regulating the expression of their target genes (1, 2). IL-6 regulates the production of acute phase proteins in hepatocytes and stimulates differentiation of B and T cells and proliferation of keratinocytes, mesangial, and myeloma/plasmacytoma cells. The IL-6 cytokine family transduces their signals via a common receptor subunit, glycoprotein 130 (gp130). In the case of IL-6, binding of the ligand to the non-signaling ␣-receptor subunit recruits the -receptor subunits, gp130, which form homodimers. The receptor dimerization leads to phosphorylation and activation of the non-receptor tyrosine kinases of the JAK family that are pre-associated with gp130. JAKs in turn phosphorylate specific tyrosine residues in the cytoplasmic portion of gp130, which serve as docking sites for signaling molecules containing Src homology 2 (SH2) domains. Six tyrosine residues are phosphorylated in gp130 upon ligand stimulation, leading to the activation of two major pathways (for viewed, see Ref.3). The four phosphotyrosine residues at the C-terminal region of gp130 serve as recruitment sites for Stat3 (4), whereas phosphorylation of its second membrane-proximal tyrosine residue leads to recruitment of SHP2 (3). SHP2 serves as an adaptor protein mediating the activation of the Ras-Rafmitogen-activated protein kinase signaling pathway. As a protein-tyrosine phosphatase, SHP2 also plays a negative regulatory role in IL-6 signaling (5, 6). The same site also mediates binding of the IL-6-induced feedback inhibitor SOCS3, which attenuates IL-6 signaling by inhibiting JAKs as well as by triggering protein degradation (7,8).The LIM domain, characterized by a double zinc finger structure, was originally identified in the homeodomain transcription factors Lin-11, Isl-1, and Mec-3, which have established roles in the central nervous system, and was subsequently found in a variety of proteins with diverse functions. The LIMcontain...
This article describes the interplay between the serine/threonine kinase maternal embryonic leucine zipper kinase (MELK) and the enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) and provides a basis for targeting this enzymatic pathway in mature natural killer/T-cell malignancies.
Background: Differentiation therapies achieve remarkable success in acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML). However, clinical benefits of differentiation therapies are negligible in non-APL AML, which accounts for the majority of AML cases. Dihydroorotate dehydrogenase (DHODH) regulates the fourth step of the de novo pyrimidine synthesis pathway. DHODH is a key therapeutic target for auto-immune diseases and cancer, particularly differentiation of AML. ASLAN003 is a novel, potent small molecule DHODH inhibitor being developed in AML by ASLAN Pharmaceuticals. Methods: We investigated activity of ASLAN003 in AML cell lines and primary bone marrow (BM) cells (NUS Leukemia Tissue Bank) from patients with AML (N = 14) or myelodysplastic syndromes (MDS) (N = 6) and healthy control (N = 1). We performed CTG assay, FACS analysis of cell viability and myeloid markers, wright-giemsa staining, NBT reduction assay, and qRT-PCR analysis of key lineage transcription factors to evaluate the effects of ASLAN003 on cell growth, differentiation, apoptosis, and gene expression changes in vitro. Two AML cell lines and 1 leukemic patient derived xenograft (PDX) line (NUS Leukemia Tissue Bank) were studied in NSG xenograft mice. Mice were administrated with vehicle control or ASLAN003 50 mg/kg by oral gavage once daily. Results: ASLAN003 inhibited leukemic cell growth of THP-1, MOLM-14 and KG-1 with IC50 of 152, 582 and 382 nM, respectively, at 48 h. Treatment of these leukemia cells with ASLAN003 for 96 h consistently resulted in remarkable increase of CD11b (p < 0.001) and displayed morphologic changes of terminal differentiation and positivity for NBT reduction. ASLAN003 was active in differentiation with an EC50 of 28, 85, and 56 nM, in these 3 lines, respectively. ASLAN003 induced approximately 2-fold higher CD11b+ cells than Brequinar (BRQ), another DHODH inhibitor. Addition of uridine rescued differentiation and improved cell viability in ASLAN003 treated-cells, implying on-target specificity of ASLAN003. Mechanistically, ASLAN003 induced differentiation through induction of myeloid lineage transcription factor Runx1, Pu.1, Gif1 and repression of HoxA9, Gata1. The response of primary BM cells to ASLAN003 was classified into 3 categories: sensitive if any of myeloid markers CD11b, CD14, CD13 or CD33 increased ≥ 15%; moderate: ≥ 5%, but < 15%; resistant: < 5%. Among AML samples, we observed 6 (43%) sensitive cases, 6 (43%) moderate cases and 2 (14%) resistant cases. Three (50%) MDS samples displayed sensitive response and 3 cases (50%) showed moderate response. The healthy control sample was resistant to ASLAN003. Importantly, ASLAN003 promoted differentiation and cell death of myeloid cells in one relapsed AML case. Morphologic analysis and NBT assay demonstrated the features of neutrophil differentiation in selected ASLAN003-treated primary AML blasts. For in vivo experiments, significantly prolonged survival was seen in ASLAN003-treated groups when compared to vehicle control group in both MOLM-14 (p = 0.031) and THP-1 (p < 0.001) xenograft models. ASLAN003 substantially reduced disseminated tumors and leukemic infiltration into liver in xenografted mice. The human CD45+ cells were significantly reduced in BM, peripheral blood, spleen and liver, with significantly increased differentiation of AML cells (CD11b and CD14 positive cells) in BM of treated mice in both models (p < 0.01). We also evaluated the therapeutic efficacy of ASLAN003 in one PDX line, AML-14. At the end of experiments (day 77 post treatment), all PDX mice were alive in both control and ASLAN003 group. The leukemic burden was significantly lower in ASLAN003-treated PDXs than in vehicle-treated PDXs (p = 0.04). Overall, these data demonstrate potent in vivo efficacy of ASLAN003 in inducing myeloid differentiation of blast cells and the drug appears highly tolerable even after prolonged administration. Conclusion: ASLAN003 is a novel, highly potent DHODH inhibitor that induces terminal differentiation, inhibits cell growth and promotes cell death of AML blasts, including relapsed AML blasts. ASLAN003 prolongs survival and shows therapeutic effects in mice bearing different AML cell lines and reduces leukemic burden in an AML PDX model. Currently, ASLAN003 efficacy is being evaluated in a Phase IIa clinical trial in patients with AML (NCT03451084; Ting, ASH abstract 2018). Disclosures Seet: ASLAN Pharmaceuticals: Employment, Equity Ownership. Ooi:ASLAN Pharmaceuticals: Employment, Equity Ownership. Lindmark:ASLAN Pharmaceuticals: Employment, Equity Ownership. McHale:ASLAN Pharmaceuticals: Employment, Equity Ownership. Chng:Amgen: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Aslan: Research Funding; Merck: Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Takeda: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Celgene: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding.
Islet1 (Isl1) belongs to the LIM homeodomain transcription factor family. Its roles in differentiation of motor neurons and organogenesis of pancreas and heart have been revealed. However, less is known about its regulatory mechanism and the target genes. In this study, we identified interactions between Isl1 and Janus tyrosine kinase (JAK), as well as signal transducer and activator of transcription (Stat)3, but not Stat1 and Stat5, in mammalian cells. We found that Isl1 not only forms a complex with Jak1 and Stat3 but also triggers the tyrosine phosphorylation of Jak1 and its kinase activity, thereby elevating the tyrosine phosphorylation, DNA binding activity, and target gene expression of Stat3. In vivo, the tyrosine-
Differentiation therapies achieve remarkable success in acute promyelocytic leukemia, a subtype of acute myeloid leukemia. However, excluding acute promyelocytic leukemia, clinical benefits of differentiation therapies are negligible in acute myeloid leukemia except for mutant isocitrate dehydrogenase 1/2. Dihydroorotate dehydrogenase catalyses the fourth step of the de novo pyrimidine synthesis pathway. ASLAN003 is a highly potent dihydroorotate dehydrogenase inhibitor that induces differentiation, as well as reduces cell proliferation and viability, of acute myeloid leukemia cell lines and primary acute myeloid leukemia blasts including in chemo-resistant cells. Apoptotic pathways are triggered by ASLAN003, and it also significantly inhibits protein synthesis and activates AP-1 transcription, contributing to its differentiation promoting capacity. Finally, ASLAN003 substantially reduces leukemic burden and prolongs survival in acute myeloid leukemia xenograft mice and acute myeloid leukemia patient-derived xenograft models. Notably, the drug has no evident effect on normal hematopoietic cells and exhibits excellent safety profiles in mice, even after a prolonged period of administration. Our results, therefore, suggest that ASLAN003 is an agent targeting dihydroorotate dehydrogenase with potential in the treatment of acute myeloid leukemia. ASLAN003 is currently being evaluated in phase 2a clinical trial in acute myeloid leukemia patients. Article Summary 1. ASLAN003, a novel, potent dihydroorotate dehydrogenase inhibitor, induces differentiation of acute myeloid leukemia cells in vitro and in vivo. 2. ASLAN003 triggers apoptosis, inhibits protein synthesis and activates AP-1 transcription.
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